Methods of chromosome analysis What is his karyotype? He is 4 years old (looks like 2 y.o.) Knows few words Inadequate reaction Abnormal face Short hands Heart abnormalities, kidney abnormalities Plurimapformative syndrome 1. Collecting of nuclear cells (just leucocytes if blood is used) 1
2. Cell culturing cells in different stages of cell cycle Thermostat Blood cells + growth broth 37º C 72 hours After 72 hours Cells in different stages of cell cycle Proliferation 3. Preparation of chromosomes for analysis Cytostatic - colchicine Hypotonic solution Cells in different stages of cell cycle Mitosis arresting Cell sedimentation Chromosome dispersing 2
4. Painting of chromosomes Painting Slide with fixed cells Slide with metaphase plates 5. Karyotype analysis Printing of picture Cutting individual chromosomes Preparation of karyotype 3
47,XY,+21 Down syndrom Cytogenetic methods used for karyotype analysis: To identify numeric aberrations analysis of metaphase chromosomes uniformly painted. To identify numeric or structural aberrations analysis of metaphase or prometaphase chromosomes using Q, G, R banding. For exact identification of chromosomes analysis of metaphase chromosomes using T or C banding. Analysis of human karyotype Metaphase chromosomes Prometapfase chromosomes Interphase chromosomes Uniform painting G,Q,R,C,T banding G, R banding X and Y sex chromatin analysis Molecular citogenetical methods FISH, mfish SKY CGH *** No of bands per haploid set: 300-400 m / 550 pm / 850p 4
Type of banding G Used dye Giemsa Q R (revers) C (centromere) Quinacrin (fluorescent) Giemsa or fluorescent Giemsa or fluorescent T (telomere) Giemsa or fluorescent 13 Chromosome staining Homogenous Banding G Q R C T Bands G+ = Q+ = R Bands G- = Q- = R + Cum se colorează diferit acelaşi crs? 5
Uniform staining 6
C banding Chromosomal banding Chromosome Arm 4p22.2 5q13.4 9p21.3 Region Band Subband 7
1p35 1p22 1q23 1q41 46,XY,del(1)(p11-p34) 46,XY,del(1)(p11-p34) 46,Y,del(X)(q12.1-q24.3) 8
Chromosome 4 Identification of X and 18 Identification of X, Y and 18 FISH in chromosomal analysis Identification of 18 of 21 Identification of 18 and 21 25 46,XX 47,XX,+21 47,XX,+21 47,XX,+21 9
SKY mfish 10
Karyotype Normal Normal polymorphisms Aneuploidy Polyploidy Chromosomal abberations 46,XX 46,XY 46,XX,9qh+ 46,XX,14s++ 47,XX,+21 45,XY,-12 48,XXXY 45,X 69,XXX 69,XXY 46,XY,16qh- 46,XX,1q- 46,XY,16p+ 46,X,r(X) 46,XY,del(5p) 46,XX,t(12,22) Normal karyotype 46,XX 46,XY Normal phenotype Polymorphisms Chrs D and G variations in ph or s: Abnormal Chrs 1,9,16,9,Y variations in qh phenotype Aneuploidies Balanced structural aberrations Unbalanced structural aberrations Trisomy 47,XXX; 47,XXY; 47,+21; 47,+13; 47,+18; 47,+8 Monosomy 45,X inv t rob del dup r i Fenotip patologic Abnormal phenotype Fenotip patologic??? Polymorphisms p and q contain coding and non-coding regions p+ orp- - abnormality q+ orq- - abnormality c longer or shorter polymorphism t longer or shorter polymorphism h+ orh- - polymorphism s+ ors- - polymorphism 11
Sex chromatin analysis X chromatin analysis = Barr body test identification of non-active X, in interphase nuclei of somatic cells Y chromatin analysis = F body test identification of 2/3 of qy in interphase nuclei of somatic cells or sperms Normal karyotype X chromosome - 1529 genes: Structural somatic genes Regulatory feminisation genes; Structural feminisation genes; Structural masculinisation genes Y chromosome - 344 genes: regulatory masculinisation genes pseudogenes!!! q arm contains constitutive heterochromatin 12
!!! Important X chromosome is mandatory for and : In 46,XX only one X is active In 46,XY are active both X and Y In 47,XXX - only one X is active In 47, XXY- are active only one X and one Y In 48, XXXY- are active only one X and one Y Y chromosome is mandatory for How to explain this? Sexual dimorphism - balanced Primary sexual traits - different Secondary sexual traits - different Somatic traits - similar 45,X 47,XXY 13
46,XX 46,XY Active X - euchromatin Inactive X - heterochromatin Active X - euchromatin Y chromosome p arm active q arm - inactive Barr body = 1μm F body = 0,25 μm 14
Barr body X sex chromatin Nr Barr = X - 1 48,XXXY 45,X 50,XXXXYY 49,XXXXX Barr body test XXX X XXXX XXXXX F body test Y YY X sexual chromatin: Barr body test - represents an inactive X, facultative heterochromatin, in somatic cells 46,XX; - represents a Barr body 1 m in diameter; -Barr body test is useful for identification of No of X chromosomes in somatic cells: No X = No Barr + 1. 46,XX 1 Barr body 46,XY 0 47,XXX 2 47,XXY - 1 45,X 0 48,XXXX 3!!! M. Lyon (1961) - explanation of Barr bod y 15
Mary Lyon hypothesis After 16 th day!!! In 46,XX cells after 16 th day of development only one X remains active In 50% of cells maternal X is active, in 50% - paternal X 46,XX 45,X active +X paternal inactive 45,X active + X maternal inactive Y sexual chromatin: F body test - represents 2/3 of q arm of Y chromosome, its a constitutive heterochromatin in somatic cells or 50% of sperms (23,Y only); - represents fluorescent F body 0,25 m in diameter; - F body test is useful for prenatal identification of gender: No Y = No F bodies 46,XX 0 46,XY 1 47,XYY 2 47,XXY - 1 48,XXYY 2 46,X,i(Yp) 0 46,X,i(Yq) 1 (0,5 m) 16